Inactivation of phage T7 by near-ultraviolet radiation plus hydrogen peroxide: DNA-protein crosslinks prevent DNA injection.

Abstract

A nonlethal concentration of H202 (0.05%) greatly enhances near-ultraviolet (NUV) inactivation of phage T7. Simultaneous treatment with H202 and NUV reduces the amount of DNA injected into the bacterial host, but not-the number of phage adsorbed. Not only were recombination and gene expression of late markers reduced upon treatment of phage '11 with NUV plus H202, but also a gradient of recombination resulted, with markers injected first reduced to a lesser extent than those injected last. Labeling Techniques. Phage used for adsorption experiments were labeled as reported (9). The method of Hawkins (11) was used in double labeling. Both procedures yielded from 10-6 to 10-7 cpm per phage.Adsorption and Injection Studies. Phage were separated by the polyethylene glycol/dextran sulfate method (12) and further purified by differential centrifugation (13) prior to gradient centrifugal studies. Titers of ca. 5 X 1012 plaqueforming units per ml were routinely obtained. For phage adsorption and injection assays (14) E. coli B was infected with treated phage; 8 min after infection of cells, chloramphenicol was added, a sample was removed to assay radioactivity, and infected bacteria were removed by centrifugation. The pellet was resuspended and an aliquot was withdrawn to determine percentage of phage adsorbed. To determine the fraction of DNA injected into the host, cells were lysed, treated with DNase I, and centrifuged to remove bacterial debris as well as whole phage particles, and the supernatant was then assayed for radioactivity. This procedure is based on the assumption that the solubilized radioactivity represents the amount of DNA that is injected into the bacterial host, because only it is available for digestion by deoxyribonuclease.Genetic Recombination. For genetic crosses between amber mutants (15, 16) E. colh 011' (Su+) was coinfected with either T7 amli (gene 8) or am28 (gene 5), and one of a series of different amber mutants (helper phage) located throughout the T7 geqome. Lysates were assayed on 011' (Su+) and B (Su-) for total progeny and wild-type recombinants, respectively. Results are expressed as percentage of normal recombination (16). Average frequencies of wild-type recombinants for the control crosses were compatible with those in previous reports (15,16).Gel Electrophoresis Studies. E. coli B was grown at 37°C to 4 X 108 cells per ml in M9 medium plus 2 mg of casamino acids per ml, irradiated with 72 J-M2 of far-UV radiation to suppress host cell protein synthesis, and incubated for 30 min at 30°C in the dark. Aliquots (2 ml) were then infected with appropriately treated T7 at a multiplicity of infection of 10 and incubated with 10 ,tCi (1 Ci = 3.7 X 1010 becquerels) of [-5S]-methionine (New England Nuclear) per ml for 24 min at 30°C. Samples were washed and concentrated 10-fold by centrifugation and applied to polyacrylamide gels. Sodium dodecyl sulfate/polyacrylamide gradient slab gels were prepared 1.5 mm thick with a linear gradient of 8-13% acrylamide and a 5% acrylamide stackin...

Exploiting radiation damage to map proteins in nucleoprotein complexes: The internal structure of bacteriophage T7

“…One candidate is gp6.7, a minor component of the nucleocapsid known to associate with the portal (Hauser et al, 2012), a possibility already proposed by Kemp and coworkers (Kemp et al, 2005). We tentatively interpret the 11 nm-long segment of radiation-resistant density as ∼ 30 bp of duplex DNA at one end of the linear T7 genome, presumably the “left” end which is the last to enter the capsid in packaging and the first to leave in infection (Black, 1989; Hartman et al, 1979). In this scenario, the DNA end is “threading the needle”, being ideally positioned to pass through the portal and the tail-tube after blockage by the axial protein is removed.…”

Biochemistry and pathology of radical-mediated protein oxidation

“…[151]), although protein damage and DNA-protein cross-linking were directly studied only to a limited extent [152]. Damage to mitochondrial electrontransport proteins by hypochlorite [153] and other agents has been studied, and noted to be enhanced by vitamin E deficiency [154].…”

Single-Molecule and FRET Fluorescence Correlation Spectroscopy Analyses of Phage DNA Packaging: Colocalization of Packaged Phage T4 DNA Ends within the Capsid

“…Among significant differences found among a number of packaged phage capsids are the following. Depending upon the phage capsid, DNA is oriented transverse (T7)11 or parallel (T4)12; 13 to the head-tail axis of the phage particle; the first DNA end packaged is the last end out (λ, T7)14; 15 or the first end packaged is the first end out (T4)16; and although packaged to comparable 500 mg/ml density, DNA is found in the capsid as a protein-free condensate (λ), found together with a discrete internal protein core structure (T7)11, or is packed together with several thousand molecules of mobile internal proteins9 (T4). These and other significant differences, such as functional DNA-containing “giant” capsids13, argue against a single deterministic packaged phage capsid DNA structure.…”